Project Abstract The majority of women with relapsed and advanced ovarian cancer (OC) have then very limited therapeutic options. Checkpoint blockade has shown objective responses in less than 15% in patients. Therefore, the development of more potent immunotherapy approaches such as chimeric antigen receptor (CAR) T cells (CAR-Ts) is critical in these patients. We have identified B7-H3 as a valid target for CAR-Ts in OC. We have generated B7-H3.CAR-Ts and successfully tested them in solid tumor models including OC models. In addition, since the B7-H3.CAR we have developed cross-reacts with mB7-H3, we conducted efficacy and safety studies in immunocompetent mice showing antitumor activity without toxicity. Having identified and validated B7-H3 as a target for OC, in this application we aim at overcoming the tumor microenvironment (TME) immunosuppression in OC to fully exploit the potential of the CAR technology. The TME in OC is characterized by a cellular network that promotes angiogenesis and shapes immunosuppressive cells. In particular, tumor associated macrophages (TAMs) and myeloid-derived suppressive cells (MDSCs) are abundant in the TME of OC, and inhibit effector T cells. As compared to T cells, NKTs possess the innate property to co-localize with TAMs and to exploit lytic effects on TAMs in a CD1d-dependent manner via their invariant TCR (iTCR). We have published and generated additional preliminary data showing that CAR-expressing human NKTs are dual specific targeting both tumor cells via CAR and TAMs via native iTCR. Furthermore, we have generated preliminary data showing that human NKTs can also be engineered to release IL-12, a cytokine known to reprogram MDSCs. We hypothesize that NKTs engineered to express the B7-H3.CAR and IL-12 will overcome critical challenges of adoptive immunotherapy of solid tumors: effector cell localization to the tumor site, selective killing of tumor cells via B7-H3.CAR, elimination of tumor-protective TAMs via CD1d engagement by the iTCR, and reprogramming of MDSCs via IL-12. Our new preliminary data also revealed that IL-12 potently enhances CD62L-associated stem-like program in NKTs likely via a novel mechanism, associated with unique ?vitamin D signature?. We thus hypothesize that human NKTs may have an intrinsic plasticity not previously appreciated, and that IL-12 may reprogram NKTs to a more immature phenotype via vitamin D pathway. Three specific Aims are proposed: Aim 1: To evaluate whether B7-H3.CAR and IL-12 engineering of NKTs and native iTCR cooperate in targeting OC cells and shaping the TME in an immunocompetent murine model. Aim 2: To mechanistically assess how IL-12 expressed by NKTs promotes NKTs with longer persistence upon adoptive transfer. Aim 3: To evaluate the antitumor activity of engineered human NKTs in Human-Immune Tumor (HIT) mice.